DNA construct comprising an FGF1B promoter region operably linked to an SV40 large T antigen encoding sequence

ABSTRACT

A transgenic, non-human mammal useful for assessing the effect of candidate chemotherapeutic drugs on the growth of brain tumors in vivo is provided. Incorporated into the genome of the transgenic mammal, which preferably is a rodent, is a transgene which comprises a promoter comprising the nuclear factor binding region of the RR 2  cis acting element of a fibroblast growth factor  1 B (FGF 1 B) promoter. Operably linked to the promoter is reporter gene comprising a sequence which encodes the SV 40  large T antigen. A transgenic, non-human mammal useful for identifying and isolating FGF 1  producing brain cells. Incorporated into the genome of these transgenic animals is a transgene which comprises a promoter comprising the nuclear factor binding region of the RR 2  cis acting element of an fibroblast growth factor  1 B (FGF 1 B) promoter. Operably linked to the promoter is reporter gene comprising a sequence which encodes a protein or polypeptide other than an SV 40  large T antigen. A method of obtaining neural stem cells from a sample of cells obtained from an animal is also provided. Such method comprises introducing the FGF 1 B-detector transgene into a sample of cells that have been obtained from the animal, and assaying for expression of the detectable marker in the cells, wherein cells that express the marker are neural stem cells. The cells which express the detectable marker can then be isolated from the population to provide a sub-population of neural stem cells.

This application claim priority to U.S. Provisional Application Ser. No.60/252,745 filed Nov. 22, 2000.

BACKGROUND

Animal model systems are useful tools for identifying and characterizingtherapeutic agents. Examples of such model systems are transgenic animalmodels with brain tumors. Currently, there are several transgenic animalmodels that recapitulate key features of human primitive neuroectodermaltumors (PNET) (Fung, K. M., and Trojanowski, J. Q., (1995) Animal modelsof medulloblastomas and related primitive neuroectodermal tumors. Areview. J. Neuropathol. Exp. Neurol. 54, 285-296). Members of each ofthese transgenic animal lines develop PNETs arising in different, yetdistinct, brain regions. However, the animals in these four transgeniclines also have some features in common, namely the expression of theneuronal cell marker synaptophysin (Fung, K. M., Chikaraishi, D. M.,Suri, C., Theuring, F., Messing, A., Albert, D. M., Lee, V. M.,Trojanowski, J. Q. (1994). Molecular phenotype of simian virus 40 largeT antigen-induced primitive neuroectodermal tumors in four differentlines of transgenic mice. Lab. Invest. 70, 114124). The presence of thiscell marker indicates that these tumors are derived from cells that havedifferentiated beyond the earliest stages of neural stem cells.

It is desirable to have additional animal models for identifying agentswhich are effective at preventing, slowing or reversing the growth ofbrain tumors. It is especially desirable to have an animal whose braintumor cells are at an early stage of differentiation, i.e., the tumorcells have not yet progressed to the stage where they are expressingmarkers that are indicative of neurons (synaptophysin andneuron-specific enolase), astrocytes (glial fibrillary acidic proteinand S-100), or oligodendrocytes (galactocerebroside).

SUMMARY OF THE INVENTION

The present invention provides a novel, transgenic, non-human mammaluseful for assessing the effect of candidate chemotherapeutic drugs onthe growth of brain tumors in vivo. Incorporated into the genome of thetransgenic mammal, which preferably is a rodent, is a transgene whichcomprises a promoter comprising the nuclear factor binding region of theRR2 cis acting element of a fibroblast growth factor 1B (FGF1B)promoter. Operably linked to the promoter is reporter gene comprising asequence which encodes the SV40 large T antigen. Such transgene isreferred to hereinafter as the “FGF1B-T antigen” transgene. The presentinvention also provides a DNA construct comprising the transgene.

The present invention provides a novel, transgenic, non-human mammaluseful for identifying and isolating FGF1 producing brain cells.Incorporated into the genome of these transgenic animals is a transgenewhich comprises a promoter comprising the nuclear factor binding regionof the RR2 cis acting element of an fibroblast growth factor 1B (FGF1B)promoter. Operably linked to the promoter is reporter gene comprising asequence which encodes a protein or polypeptide other than an SV40 largeT antigen. Such protein is a detectable marker that permitsidentification and separation of transgenic animal brain cells that areexpressing such marker from transgenic animal brain cells that are not.Such transgene is referred to herein after as the “FGF1B-detector”transgene.

In one embodiment, nuclear factor binding region of the RR2 cis actingelement is derived from the human FGF1B promoter and comprises thesequence 5′ ACCTGCTGTTTCCCTGGCAACTC, 3′, SEQ ID NO. 1. In oneembodiment, the promoter comprises nucleotide −540 through nucleotide +1of the human FGF1B promoter. In another embodiment the promoter is achimeric promoter which comprises SEQ ID NO. 1 and the minimal HerpesSimplex Virus (HSV) thymidine kinase (tk) promoter.

The transgenic animals whose genome comprises the FGF1B-T antigentransgene exhibit a phenotype which is different from the phenotype ofnormal animals of the same species. For example, these transgenic micewhose genome comprises the FGF1B-T antigen transgene develop tumor fociin the pontine gray area of the brain The brain tumor cells that arederived from mature transgenic mice (i.e., transgenic mice that aregreater than 3 months old) whose genome comprises the FGF1B-T antigentransgene lack terminal differentiation markers for astrocytes (glialfibrillary acidic protein and S-100) or neurons (synaptophysin andneuron-specific enolase). The brain tumor cells of the transgenic micewhose genome comprises the FGF1B-T antigen transgene also express higherlevels of proliferating cell nuclear antigen and vimentin than braincells from normal mice at the same stage of development, indicating thatthe brain tumor cells of such transgenic mice are proliferative.

The present invention also provides isolated mutant non-human mammalianzygotes whose genome comprises the present transgenes. Such zygotes areuseful for making the mutant non-human mammals.

The present invention further relates to isolated transformed braincells derived from the transgenic animals whose genome comprises theFGF1B-T antigen transgene and from the transgenic animals whose genomecomprises the FGF1B-detector transgene. Such brain cells are useful foridentifying markers that are unique to neural stem cells. Neural stemcells are considered possible therapeutic agents for treating patientswith neurodegenerative diseases such Alzheimer's disease, Parkinson'sdisease, stroke, and spinal cord injury.

The present invention also relates to a method of using the transgenicanimals whose genome comprises the FGF1B-T antigen transgene to identifydrugs which inhibit the growth of brain tumors in vivo.

The present invention also provides a method of obtaining neural stemcells from a sample of cells obtained from an animal. Such methodcomprises introducing the FGF1B-detector transgene into a sample ofcells that have been obtained from the animal, and assaying forexpression of the detectable marker in the cells, wherein cells thatexpress the marker are neural stem cells. The cells which express thedetectable marker can then be isolated from the population to provide asub-population of neural stem cells. Preferably, the sample is a braintissue sample.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Restriction map of FIB-Tag construct. The thick line representsthe human FGF-1B promoter sequence. The hatched box represents the SV40immediate early genes. Dashed lines indicate the introns for the large Tand small t antigens. ATG and TAA are the translational initiation andtermination codons for SV40 T antigen. The polyadenylation site issymbolized by An. Dotted lines represent the vector sequence derivedfrom pGL2-Basic plasmid. As such, an additional copy of the SV40polyadenylation signal sequence is located upstream from the FGF1promoter to prevent stray transcription initiated from flanking hostgenomic sequences.

FIG. 2. Southern blots of F1B-TAg transgenic mouse DNA. Mouse-tail DNAwas digested with BamHI, separated on an agarose gel, and blotted to afilter. The filter was hybridized to a 1.2-kbp HindIII fragment derivedfrom SV40 immediate early gene sequences. Each of the four lines has adistinct pattern of hybridization, indicating different integrationsites of the transgenic DNA. Panel A shows the offspring of mice 98-29,98-25 and 88-11. Panel B shows the offspring of mouse 94-8. Lambda DNAdigested with HindIII was used as a molecular weight marker and thesizes in kbp are indicated on the left of each panel.

FIG. 3. A pedigree of the four FIB-Tag transgenic lines, 88, 94H, 94Land 98. A partial pedigree analysis is presented to examine theheritability of the phenotype of premature death and brain tumor.Females are indicated by open circles, males by open squares, andtransgenic mice by solid circles and squares. The founder 94 containstwo non-allelic transgenes. Hatched circles and squares indicate thosethat inherited the low copy number locus in the 94L line. The mouse94-8-82 contains only the high copy number locus (94H), while the mouse94-16 contains only the low copy number locus (94L).

FIG. 4. (A) Cresyl fast violet-stained sagittal section showing a largefocal tumor in the caudal pons of a 94H strain mouse at P 147. Thecerebellum is flattened and anterior lobules 1-6 are somewhat agranular.(B) A hematoxylin and eosin-stained sagittal section showing multifocaltumors (arrows) in the striatum and thalamus of an adult (P224) 94Lstrain mouse. (C) A sagittal section from a 98 strain mouse (P 166)labeled with an SV40 T antibody, counter-stained with cresyl fastviolet, showing tumor foci distributed in a diagonal stream (arrows),from the tegrnentum to the interpeduncular nucleus. (D) Highmagnification photomicrograph of a vimentin-stained tumor with smallercell clusters (arrows) at the periphery. A sagittal section (E) throughthe cerebellum and pons of a 94H mouse (P 172). This animal is unusualin that cerebellar lobules 1 and 2 are invaded by proliferating cells(small arrows) organized in perpendicular arrays from the pial membranethrough the molecular layer. These ectopic cells may be following majorblood vessels, which are organized similarly. The main tumor (tm) liesventral to cerebellar lobule 1. (F) The ependymal membrane (ep) adjacentto the dorsal tegmental nucleus, is thickened by proliferating cells,and is heavily vascularized (arrows). BS, brain stem; cb, cerebellum;ct, cortex; ep, ependymal membrane; g, granule cell layer; h,hippocampus; ic, inferior colliculus; ip, interpeduncular nucleus; m,molecular layer; P, Purkinje cell layer; st, striatum; tg, tegmentalarea; th, thalamus; tm, tumor; IVV, fourth ventricle; LV, lateralventricle; 1,6, cerebellar folia 1 through 6. Scale bars: A, E, 500 μm;B, C, 250 μm; D, F, 100 μm.

FIG. 5. A schematic of the distribution of brain tumors in 88, 98, 94Hand 94L transgenic lines.

FIG. 6. A cresyl fast violet-stained sagittal section (A) through themidline of a P25 mouse brain (88 line), showing pigmented cells at thesite of tumor origin in the pontine raphe/dorsal tegmental nucleus, justbelow the ependymal membrane at the rostral surface of the fourthventricle. In adjacent sections, proliferating cells are (B) SV-40T, (C)GFAP and (D) vimentin positive. cb, cerebellar cortex; dtg, dorsaltegmental nucleus; ep, ependymal membrane; IVV, fourth ventricle. Scalebars: 100 μm.

FIG. 7. Sagittal sections through the brain stem of an adult (P200)mouse of the 88 line. (A) A cresyl fast violet-stained section showing alarge tumor extending throughout the dorsal and ventral tegmental nuclei(tg) and pontine raphe nucleus, and into the fourth ventricle. Aseparate tumor is located in the interpeduncular nucleus (ip) at theventral surface of the brain stem. High levels of punctate PCNA stainingare detected throughout the tumor (B); the vimentin antibody isdistributed similarly (C). In a mature animal, GFAP staining (D) ispresent at the periphery of the tumors and throughout tracts (smallarrows) joining the two major foci; however, it is largely absent fromthe body of the tumor. All tumors were FGF1-negative (E) and SV40positive (F). cb, cerebellar cortex; ic, inferior colliculus; ip,interpeduncular nucleus; om, occulomotor nucleus; tg, tegmental nuclei;.Large arrows in E and F indicate the ventral border of the tumor. Scalebars: A-D 500 μm; E-F, 250 μm.

FIG. 8. Sagittal sections through the tegmental region of an adult 88mouse showing the absence of (A) neuron specific enolase, (B)synaptophysin, and (C) S-100-positive cells within the tumor. cb,cerebellar cortex; ic, inferior colliculus; large arrows indicate theborder of the tumor; small arrows in B and C indicate a few remaininglabeled cells. Scale bars: A, 100 μm; B, C, 200 μm.

FIG. 9. All major foci are heavily vascularized. Panel (A), vWF-positivecells associated with blood vessels at the tumor periphery. (B) Bloodvessels within the tumor lined with vWF-positive cells. Larger bloodvessels are also positive for CD31 (C) and VEGF (D). Arrows indicateblood vessels. Scale bars: A, 200 μm; B, 100 μm; C, D, 50 μm.

FIG. 10 is the nucleotide sequence (SEQ ID NO. 2) of the plasmidcontaining the transgene designated FIB(−540)-Tag. Nucleotides 1-594 arederived from the human FGF1B promoter (Accession No. Z14150 cloned inour laboratory. Nucleotides 595-3233 are derived from the reversecomplement of SV40 from nucleotides 5171-2533 (Accession No. NC-001669).

Nuleotides 3234-36087 are derived from nucleotides 2544-5597 of thepGL2-Basic vector (Promega Corp).

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides a transgenic, non-humanmammal useful for assessing the effect of a candidate chemotherapeuticagent on the growth of brain tumors.

Incorporated into the genome of the transgenic mammal is the FBF1B-Tantigen transgene which comprises a promoter comprising an activeportion of the FGF1B promoter. Operably linked to the promoter is areporter gene, i.e., DNA fragment, comprising a sequence encoding theSV40 large T antigen. The term “active portion of the FGF1B promoter” asused herein refers to the nuclear factor binding region of the RR2 cisacting element of the FGF1B promoter. In one embodiment the activeportion is derived from the human FGF1B promoter and comprises SEQ IDNO. 1.

In another aspect, the present invention provides a non-human,transgenic mammal, preferably a transgenic rodent, useful foridentifying and providing FGF1 producing brain cells. In accordance withthe present invention, it is expected that FGF1 producing brain cellsare neural stem cells. Incorporated into the genome of these transgenicanimals is the FGF1B-detector transgene which comprises a promotercomprising the nuclear factor binding region of the RR2 cis actingelement of an FGF1B promoter. Operably linked to the promoter is areporter gene comprising a sequence which encodes a protein other thanan SV40 large T antigen. Such protein is a detectable marker thatpermits identification and isolation of transgenic animal brain cellsthat are expressing such marker from transgenic animal brain cells thatare not.

The term “mammal” as used herein refers to any non-human mammal. Suchanimals are, for example, rodents, non-human primates, sheep, dogs,cows, and pigs. The preferred non-human mammals are selected from therodent family including rat and mouse, more preferably mouse. A“transgenic mammal” as used herein refers to an animal containing one ormore cells bearing genetic information, received, directly orindirectly, by deliberate genetic manipulation at the subcellular level,such as by microinjection or transfection with recombinant DNA, orinfection with recombinant virus. The term “germ cell-line transgenicanimal” refers to a transgenic animal in which the genetic informationwas introduced into a germ line cell, thereby conferring the ability totransfer the information to offspring. If such offspring in fact possessthe transgene, they too are transgenic mammals.

PROMOTER

In one aspect, the promoter may comprise in order (i) the RR2 cis actingelement (nucleotides −507 to −467 of the human FGF1B promoter), (ii) theRR1 cis acting element (nucleotides −145 to −114 of the human FGF1Bpromoter), and (iii) the proximal promoter (nucleotides −92 to −49 ofthe human FGF1B promoter), of a native or naturally occurring FGF1Bpromoter that is derived from an animal, such as for example a human, amouse, a bovine animal, a chick or an amphibian. The sequence of theFGF1B promoter of human is provided in GenBank Accession No. Z14150,which is specifically incorporated herein by reference. The sequence ofthe FGF1B promoter of the mouse is provided in GenBank Accession No.U67609 (SEQ ID NO. 3), which is specifically incorporated herein byreference. The RR2 region of mouse FGF1B promoter contains a minimalsequence from nucleotide 640-679 of Accession No. U67609, based on itshomology with the human FGF1B promoter and its relative location to thetranscription start site of the mouse FGF1B mRNA.

In one embodiment, the promoter is derived from a human and comprisesnucleotides −145 to nucleotide +1, preferably nucleotide −507 throughnucleotide +1, and more preferably nucleotide −540 to nucleotide +1,which is the transcription initiation site, of the human FGF1B promoter.Preferably, the promoter which is derived from FGF1B promoter of ananimal further comprises nucleotides downstream from the transcriptionsite, such as for example the 31 nucleotides of the 5′-untranslatedsequence of the corresponding FGF1 gene.

In accordance with the present invention, it has been determined that apromoter comprising 540 base pairs of the human FGF1B promoter sequenceand the first 31 base pairs of the 5′-untranslated sequence of the humanFGF1B mRNA is particularly well-suited for preparing the transgene.

Alternatively, the promoter is a chimeric promoter which comprises theproximal promoter of a heterologous, i.e., a non-FGF1B, promoter and thenuclear factor binding region of the RR2 cis acting element of amammalian FGF1B promoter. The sequence that constitutes the proximalpromoter provides basal level of transcriptional activity. In oneembodiment, the element is derived from nucleotide −484 throughnucleotide −467 of the human FGF1B promoter and comprises SEQ ID NO. 1.In one embodiment, the heterologous promoter is the full length HSV tkpromoter (from nucleotides −200 to +67), the sequence of which isprovided in Accession No. V00467, which is specifically incorporatedherein by reference. In another embodiment, the heterologous promoter isminimal HSV tk promoter fragment which comprises nucleotide −81 throughnucleotide +67 of the full-length HSV tk promoter. Examples of otherheterologous promoters include, but are not limited to, the adenoviralE1b TATA promoter, the human cytomegalovirus major immediate-early genepromoter, the heat shock protein (hsp70) promoter, and the dihydrofolatereductase (dhfr) promoter.

REPORTER GENES

The transgene further comprises a reporter gene. As used herein the term“reporter gene” refers to a DNA fragment that encodes an assayableproduct The assayable product is a protein or peptide which permitsdifferentiation of transformed brain cells which express such gene fromcells that do not. As used herein the term “transformed” brain cellrefers to a brain cell whose phenotype is different from a normal braincell in that the transformed brain cell comprises a protein orpolypeptide that is not found in the normal brain cell. In oneembodiment, the reporter gene is derived from the SV40 genome andencodes the SV40 large T antigen. Preferably the SV40 DNA fragmentcomprises nucleotides 5163-2691, more preferably 5171-2533 of the SV40immediate early gene which is depicted in GenBank Accession No.NC_(—)00166.

In other embodiments, the reporter gene is not derived from the SV40genome. Examples of such reporter genes include, but are not limited to,genes for drug and metabolite selection of transformed cells, genes thatexpress an exogenous cell membrane protein, and genes that encode afluorescent protein, and genes that encode a protein that can bedetected by immunocytochemistry. Some examples of reporter genes thatencode proteins that permit drug or metabolite selection are the gptreporter gene, the neomycin reporter gene, the thymidine kinase reportergene, the dhfr reporter gene, the hygromycin reporter gene, thetryptophan reporter gene and the histidine reporter gene. Expression ofgpt reporter protein resistance to the HAT (hypoxanthine, aminopterinand thymidine) selection medium or to mycophenolic acid ((Mulligan R C,Berg P: Selection for animal cells that express the Escherichia coligene coding for xanthine-guanine phosphoribosyl transferase. Proc. Natl.Acad. Sci. USA 78: 2072-2076, 1981). Expression of neo confers G418resistance; while expression of tk reporter protein provides a negativeselection in the presence of gangcyclovir (Mansour S L, Thomas K R,Capecchi M R. Disruption of the proto-oncogene int-2 in mouseembryo-derived stem cells: a general strategy for targeting mutations tonon-selectable genes. Nature. 336: 348-352, 1988). Cells expressing dhfrare selected for increased resistance to methotrexate (Wigler M, PeruchoM, Kurtz D, Dana S, Pellicer A, Axel R, Silverstein S: Transforming ofmammalian cells with an amplifiable dominant-acting gene. Proc. Natl.Acad. Sci. USA 77:3567-3570, 1980). Expression of hygro provides forhygromycin B resistance (Yang Z, Korman A J, Cooper J, Pious D, AccollaR S, Mulligan R C, Strominger J L. Expression of HLA-DR antigen in humanclass II mutant B-cell lines by double infection with retrovirusvectors. Mol Cell Biol 7: 3923-3928, 1987). Expression of the trpB geneof Escherichia coli, which encodes the beta subunit of tryptophansynthase, allows mammalian cell survival and multiplication in mediumcontaining indole in place of tryptophan (Hartman S C, Mulligan R C: Twodominant-acting selectable markers for gene transfer studies inmammalian cells. Proc. Natl. Acad. Sci. USA 85:8047-8051, 1988). ThehisD gene of Salmonella typhimurium encodes histidinol dehydrogenase,which catalyzes the oxidation of L-histidinol to L-histidine. In mediumlacking histidine and containing histidinol, only mammalian cellsexpressing the hisD product survive (Hartman S C, Mulligan R C: Twodominant-acting selectable markers for gene transfer studies inmammalian cells. Proc. Natl. Acad. Sci. USA 85:8047-8051, 1988).

Reporter genes encoding a cell membrane protein, such as CD2, canfacilitate the selection of transformed brain cells by animmunoselection “panning” procedure with an antibody reacting to theexpressed exogenous membrane protein (Seed B, Aruffo A. Molecularcloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapidimmunoselection procedure. Proc. Natl. Acad. Sci. USA 84: 3365-3369,1988.)

Transformed brain cells that express a fluorescent protein can beisolated using fluorescence activated cell sorter as described by Roy etal. (Roy N S, Benraiss A, Wang S, Fraser R A, Goodman R, Couldwell W T,Nedergaard M, Kawaguchi A, Okano H, Goldman S A. Promoter-targetedselection and isolation of neural progenitor cells from the adult humanventricular zone. J Neurosci Res. 59:321-331, 2000). Examples of suchfluorescent protein include, but are not limited to luciferase protein(Myers, R. L., Ray, S. K., Eldridge, R., Chotani, M. A., and Chiu, I.-M. (1995). Functional characterization of the brain-specific FGF-1promoter, FGF-1.B. J. Biol. Chem. 270, 8257-8266.) and green fluorescentprotein (Heim R, Tsien R Y: Engineering green fluorescent protein forimproved brightness, lower wavelengths and fluorescence resonance energytransfer. Curr. Biol. 6:178-182, 1996).

The transgene also contains a ribosome binding site for translationinitiation and a transcription terminator.

In another aspect, the present invention relates to a DNA constructcomprising the transgene. Such construct is an expression vector,preferably a plasmid which allows for preparation of large amounts ofthe transgene. In such a plasmid, the transgene is flanked byrestriction sites and, preferably comprises an origin of replication.Such construct may be made by cloning the promoter sequence into avector comprising the reporter gene sequence or by cloning the reportergene sequence into a vector comprising the promoter sequence, usingconventional recombinant techniques. The DNA sequence encoding thepromoter is incorporated into the construct in appropriate frame withthe reporter gene sequence such that induction of the promoter causesexpression of the reporter gene.

In another aspect the present invention relates to a zygote or embryonicstem cell whose genome comprise the transgene. A DNA construct whichcomprises the present transgene may be integrated into the genome of thetransgenic animal by any standard method such as those described inHogan et al., “Manipulating the Mouse Embryo”, Cold Spring HarborLaboratory Press, 1986; Kraemer et al., “Genetic Manipulation of theEarly Mammalian Embryo”, Cold Spring harbor Laboratory Press, 1985;Wagner et al., U.S. Pat. No. 4,873,191, Krimpenfort et al U.S. Pat. No.5,175,384 and Krimpenfort et al., Biotechnology, 9: 88 (1991), all ofwhich are incorporated herein by reference. Preferably, the DNA fragmentis microinjected into pronuclei of zygotes of non-human mammaliananimals, such as mice, rabbits, cats, dogs, or larger domestic or farmanimals, such as pigs. These injected embryos are transplanted to theoviduts or uteri of pseudopregnant females from which founder animalsare obtained. The founder animals (Fo)founder, are transgenic(heterozygous) and can be mated with non-transgenic animals of the samespecies to obtain F1 non-transgenic and transgenic offspring at a ratioof 1:1. A heterozygote animal from one line of transgenic animals may becrossed with a heterozygote animal from a different line of transgenicanimals to produce animals that are heterozygous at two loci. Animalswhose genome comprises the transgene are identified by standardtechniques such as polymerase chain reaction or Southern assays.

The heterozygote offspring in the F1 generation or F2 generation developbrain tumors whose cells possess a neural stem cell phenotype as they donot express the markers for either glial cells or neuronal cells.Accordingly, the heterozygous transgenic animals are useful tools forscreening candidate agents capable of inhibiting, slowing or reversingthe growth of brain tumors in a mammal. Such transgenic animals are alsouseful for studying tumorigenesis in the central nervous system.

Suitable embryonic stem cells are those that have the ability tointegrate into and become part of the germ line of a developing embryo.Introduction of the transgene into the embryonic stem cell can beaccomplished using a variety of methods well known in the art, such asfor example, retrovirus-mediated transduction, microinjection, calciumphosphate treatment, or, preferably, electroporation. Thereafter, thetransgene is integrated into the genome of some of the transfectedcells, typically by non-homologous recombination. If the constructfurther comprises an antibiotic resistance gene, the transfected cellsare cultured in the presence of the antibiotic. If the construct furthercomprises a sequence encoding an assayable enzyme, the substrate for theenzyme can be added to the cells under suitable conditions, and thecells containing the product of enzymatic activity identified. Anotherassay is a Southern blot of the transfected cells genomic DNA can beprobed with a sequence designed to hybridize with the transgene. Themutant cells containing the transgene are used to prepare the mutantanimals, typically by insertion into an embryo of the same species ofanimal.

In another aspect, the present invention provides a method foridentifying agents which are effective at inhibiting, slowing orreversing the growth of brain tumors in a mammal. The method comprisesthe steps of administering the candidate agents to the transgenic animaland monitoring the growth of the tumor. Preferably, varying doses of thecandidate agents are introduced into the separate transgenic mammalsintracerebrally or by conventional modes of injection, such as forexample, by intravenous injection or intraperitoneal injection

The present invention also relates to tumor cells or tumor cell linesderived from the transgenic animal whose genome comprises the FGF1B-Tantigen transgene. Such cells are obtained from the brain tumors of suchtransgenic animals using conventional methods known in the art. In viewof their phenotype, it is expected that such cells would be useful forneural stem cell therapy for patients with neuro-degenerative diseasessuch as Alzheimer's disease, Parkinson's disease, stroke, and spinalcord injury. Such cells would also be useful for identifying additionalneural stem cell markers.

In one embodiment, the tumor cells are from the mouse brain tumor cellline KT-98, which has been deposited with the with the American TypeCulture Collection, 10801 University Blvd, Manassas, Va. 201110-2209 andassigned designation number PTA-3661. The ATCC is a Budapest Treatydepository. The transgene which is incorporated into the cells of thiscell line comprises nucleotides −540 to +31 of the human FGF1B promoterand the SV40 T antigen Tag.

In another aspect, the present invention comprises a method of obtainingneural stem from a sample of cells, e.g., a biopsy specimen, obtainedfrom an animal. The cells which are isolated from the sample usingstandard techniques are grown in tissue culture medium TheFGF1B-detector transgene is then introduced into the cultured cells, forexample by transfection or electroporation with a construct containingthe FGF1B-detector transgene, or infection with recombinant virusescontaining the FGF1B-detector transgene. The cells that haveincorporated the transgene are either expressing or not expressing thetransgene. Cells that express the transgene are the FGF-1 producingcells, and therefore neural stem cells. These neural stem cells can beisolated from the population of cells in the sample using the properdetection method, either by drug/metabolite selection, fluorescenceactivated cell sorting, immunodetective “panning,” orimmunohistochemistry. These FGF1-producing neural stem cells could thenbe propagated for stem cell based therapy in patients withneurodegenerative diseases. In one embodiment the recipient cells forthe FGF1B-detector transgene are brain cells. In another embodiment, therecipient cells for the FGF1B-detector transgene are obtained from asample other than a brain tissue sample. The recipient cells could beembryonic stem cells, bone marrow, or other types of cells, which may beused as pools of candidate cells to select for FGF1-producing cells.

The following examples are for purposes of illustration only and are notintended to limit the scope of the claims which are appended hereto.

EXAMPLE 1 A Transgene Comprising a Portion of the Human FGF1B Promoterand the Immediate Early Gene of the SV40 Genome

A. Nucleotides −540 to +31 of human FGF-1B and nucleotides 5171 to 2533of SV40 immediate early genes were cloned into the SmaI-BamHI sites ofpGL2-Basic. The T antigen coding sequence (5177 to 2533) was excisedfrom the pW2 plasmid (Chang L S, Pater M M, Hutchinson N I, di MayorcaG. Transformation by purified early genes of simian virus 40. Virology.133: 341-353, 1984) provided to us by Dr. L. S. Chang of Department ofPediatrics, Ohio State University. The enhancer sequence commonly knownas the 239-bp NcoI-PvuII fragment, extending from nucleotides 37 to 275of the SV40 genome, is not included in our construct. The enhancersequence has been shown to dictate the T antigen expression and theconcomitant tumor formation in many tissues of the transgenic animalsand is excluded to provide for tissue specificity. (Messing A, Chen H Y,Palmiter R D, Brinster R L. Peripheral neuropathies, hepatocellularcarcinomas and islet cell adenomas in transgenic mice. Nature 316:461-463, 1985).

The sequence of nucleotides −540 to +31 of FGF-1B promoter (SmaI toHindIII), is provided described in publication accession no. (Z14150)and FIG. 6 (Myers, R. L., Payson, R. A., Chotani, M. A., Deaven, L. L.,and Chiu, I. -M. (1993). Gene structure and differential expression ofacidic fibroblast growth factor mRNA: identification and distribution offour different transcripts. Oncogene 8, 341-349). The sequence ofnucleotides 5171 to 2533 (HindIII to HindIII) in the genome) is depictedin GenBank Accession No. NC_(—)001669. The sequence of the entireplasmid comprising the resulting transgene is shown in FIG. 10.

EXAMPLE 2 Transgenic Mice Comprising the Transgene of Example 1.

FGF1B promoter (nucleotides −540 to +31) was ligated upstream of theSV40 immediate early gene sequence. The resultant F1B-Tag plasmid DNA(FIG. 1) was linearized at the unique BamHI site and microinjected intozygotes. Embryos were implanted into surrogate mother mice at The OhioState University Transgenic Facilities. The transgenic mice wereidentified by PCR analysis using genomic DNA isolated from their tailsas templates (data not shown). Those that were positive with a 312-bpamplicon were subjected to Southern blotting analysis to verify thepresence of the FIB-Tag transgene.

Three founder mice (88, 94, and 98) were initially identified and mated.Representative Southern blots of the F2 generation of each of the threefamilies are shown in FIG. 2. The filter was hybridized to a 1.2-kbpHindIII fragment derived from the SV40 immediate early gene region. FIG.2A shows the offspring of F1 transgenic mice 98-29, 98-25 and 88-11.Each family had a unique pattern of hybridizing bands, indicating adistinct transgenic DNA integration site. In contrast, two differenthybridization patterns for offspring of mouse 94-8 were detected (FIG.2B) Mice 94-8-84 and 94-8-86 showed higher intensity signals than mice94-8-87, 94-8-90 and 94-8-91, indicating that the former contains ahigher copy number of the transgene. Mice with the higher copy numberalso had an additional band at 10 kbp (FIG. 2B), which helped toidentify mouse 94-8-82 as a high-copy number animal. The apparent lowintensity of hybridizing bands in this lane was due to the loading ofless DNA, which was visualized by staining the gel with ethidiumbromide. The results suggest that founder 94 had two non-allelicintegration sites (denoted H and L for high- and low-copy number,respectively).

A partial pedigree of each of the three families, showing that thetransgene was inherited according to Mendel's law, is depicted in FIG.3. The transgenic offspring of 94-8 showed the inheritance of either theH or L locus, suggesting that 94-8 inherited both the H and L loci fromher founder father. All six transgenic offspring of 94-16, as well asthose up to generation F5, inherited only the L locus (data not shown).Similarly, all five transgenic offspring of 94-8-82, as well as those upto the F5 generation, inherited only the H locus (data not shown). Thus,we concluded that the two integration sites in founder 94 have beensegregated, and the two lines are referred to as 94H and 94L,respectively (FIG. 3).

The life span of transgenic animals was considerably shorter than thatof normal mice of the parent strain. The mean (±standard deviation)survival time across four generations, excluding mice sacrificed forearly data points, was 183(±38), 178(±22), 166(±25) and 212(±35) daysfor 88, 98, 94H and 94L mice, respectively. Older animals appearedhunched and lethargic, and some animals were ataxic.

Although tumors were occasionally observed in other organs, such as thepancreas (Table I), each of the four transgenic lines manifested braintumors with complete penetrance. Within the central nervous system, theapparent sites of tumor origin were restricted to the brain stem,generally sparing the cerebral cortex, hippocampus, cerebellum andspinal cord. Tumors were comprised of small cells with scanty cytoplasmand regular round to ovoid nuclei that were stained at moderateintensity by cresyl fast violet (FIG. 4A) and hematoxylin (FIG. 4B),clearly delineating the tumors from surrounding tissue. Tumors ofdifferent sizes were distributed along myelinated fiber tracts,suggesting that infiltrating tumor cells followed these structures. Instrain 88 and 98 mice, foci were entirely restricted to the caudalpontine regions of the brain stem, and appeared to originate in thedorsal tegmental region, at the rostral surface of the fourth ventricle.In general, tumor foci in the 98 strain were distributed over a largerarea than those of line 88 animals. As with the 88 and 98 lines, majorfoci were also concentrated in the tegmental region in adult mice of the94H and 94L lines (FIG. 4C,D). Additional smaller foci were scattered ina diagonal stream from the pontine central gray into the interpeduncularnucleus at the ventral surface of the brain stem. However, unlike 88 and98 mice, tumor cells were also present in all subnuclear regions of thethalamus, and in the striatum (caudate, putamen, accumbens), ventralforebrain (olfactory tubercle, amygdala) (FIG. 4A,B), and olfactorybulbs. (data not shown). Foci were distributed differentially in 94L and94H mice: In 94L animals, tumors in the pontine gray-interpeduncularstream were far smaller and less frequent than in 94H animals, whiletumors in the thalamus, striatum, forebrain and olfactory bulbs weremore numerous and far larger. Although the degree to which the tumorsinvaded the brain stem varied across the four lines, within each linethe place of origin and final distribution of major foci were highlyreproducible across animals. The distribution of CNS tumors in eachtransgenic line is schematized in FIG. 5, and their frequency is shownin Table II.

Abnormalities were also observed in specific brain regions apparentlyunaffected by tumor activity. Although the cerebellum was generallyspared, it was frequently flattened and lobules 1 through 6 weresomewhat agranular (FIG. 4A). This was likely the result of increasedcerebellar compression between the enlarging tumor and the cranium,possibly accounting for the ataxia observed in some of the older mice.In one anomalous 94H F3 generation mouse (94-8-82-28), tumor cells,apparently associated with the ependymal lining of the fourth ventricle,extended from the dorsal surface of the medulla into posteriorcerebellar folia 1 and 2 (FIG. 4E). These cells infiltrated tangentiallythrough the molecular and Purkinje cell layers into the granule celllayer. In addition, in some animals patches of ependymal lining at therostral ventricular surface appeared thickened and distorted, extendinginto the underlying brain tissue accompanied by enlarged blood vessels(FIG. 4F). Clustered tumor cells within the Virchow-Robin spaces and. inthe subependymal region suggested that these are highly motile cellsthat migrate in association with these structures, mimicking thesecondary structures of Scherrer. In older mice (P 160 onward), theventricles were greatly distended and several mice exhibited obstructivehydrocephalus. In several 94H mice, irregularly shaped tangles ofependymal lining were present within the fourth ventricle (data notshown).

Immunocytochemical markers known to be present in neurons, glia,vascular endothelial cells, tumor cells, and proliferating cells (TableIII) were used to identify the location and cell type from which thetumors originated, and the magnitude of the proliferating population. Anantibody to the SV40 T antigen identified the smallest cellularaggregates, which at early time points (P26-30) were undetectable usingstandard histological procedures. In each of the four transgenic lines,scattered small T antigen-positive cells became detectable in thepontine raphe and dorsal tegmental nuclei by postnatal day (P)26 (FIG.6A,B). By P30, additional small aggregates were present throughout thepontine central gray (sphenoid, and dorsal and ventral tegmentalnuclei), radiating rostroventrally and mediolaterally into the pons andmedulla. These smaller foci followed the general pathways of thedecussation of the superior cerebellar peduncle, the mammillotegmentaltract, and associated blood vessels. Tumor foci in P26-P35 micecontained moderate levels of GFAP, and lower levels of vimentin (FIG.6C,D). As the animals matured, many of the smaller foci became fusedinto multicellular masses of variable size, spreading from the pontinecentral gray into the interpeduncular nucleus at the ventral pontinesurface (FIG. 7A). Punctate PCNA staining (FIG. 7B), indicatingproliferating cells in the GI and S phases of the cell cycle, becameincreasingly intense. In mature mice, high levels of vimentin werepresent (FIG. 7C), but GFAP staining was generally absent within theperimeter of all major foci (FIG. 7D), thus reversing the stainingpattern observed in the younger animals (Table III). All tumor foci werenegative for FGF1 (FIG. 7E), but continued to exhibit high levels of Tantigen (FIG. 7F).

Neuronal cell markers, including neuron-specific enolase (NSE) andsynaptophysin, and a glial cell marker, S-100, although present withintheir appropriate cell types in areas unaffected by tumor, were eithercompletely undetectable or present only in scattered cells within thetumor boundary, probably in entrapped, non-neoplastic cells (FIG. 8A-C).Enlarged blood vessels, identified by endothelial cell markers CD-31 andvon Willebrand factor (Takahashi, K., Mulliken, J. B., Kozakewich, H.P., Rogers, R. A., Folkmnan, J., and Ezekowitz, R. A. (1994) Cellularmarkers that distinguish the phases of hemangioma during infancy andchildhood. J. Clin. Invest. 93, 2357-2364), invaded all larger tumorfoci (FIG. 9A-C). For a tumor to expand beyond a prevascular size, itmust produce angiogenesis stimulators (Hanahan, D., and Folkman, J.(1996). Patterns and emerging mechanisms of the angiogenic switch duringtumorigenesis. Cell 86, 353-364). Vascular endothelial growth factor(VEGF), a potent angiogenic factor (Risau, W. (1997). Mechanisms ofangiogenesis. Nature 386, 671-674), is expressed in blood vessels withinFIB-Tag tumors (FIG. 9D).

We have generated four lines of transgenic mice using the brain-specificFGF-1B promoter to drive the expression of SV40 large T antigen, whichmanifests its transforming potential through inactivation of two majorcellular tumor suppressor proteins, p53 and pRb (Symonds et al., 1994).Each of the transgenic mice developed brain stem tumors of varioussizes, most consistently in the caudal pons. Within this region, thelocation of F1B-Tag tumors was highly reproducible, in that all fourlines developed tumors at the anterior surface of the fourth ventriclenear the midline, a region known for its susceptibility to tumorformation. Southern blotting analysis showed that the copy numbers ofthe four transgenic lines ranked as follows: 94H>98>88>94L (FIG. 2). Thedosage of the transgene is reflected in the size of the pontine tumorsformed in each line. Thus, 94H and 98 exhibited larger tegmental tumorsthan 88 mice, which, in turn, had more extensive tumors than 94L animals(FIG. 5). Furthermore, the higher copy number also reflected shortersurvival time. These results suggest that the amount of T antigen ineach of the transgenic lines is directly proportional to the severity ofthe brain tumor. It is noted that the tumor was more widely spread inthe 94H and 94L lines than in the 88 and 98 lines, often extendinganteriorly into the thalamus, striatum and olfactory bulbs (FIG. 5).Therefore, the integration sites of the transgene may also contribute tothe spatial distribution of the brain tumor.

Histogenesis of the Pontine Tegmental Nuclei

The nervous system evolves from a layer of stratified multipotent stemcells in the primitive neuroectoderm (Davis, A. A., and Temple, S.(1994) A self renewing multipotential stem cell in embryonic ratcerebral cortex. Nature 372, 263-266). Normal cells undergo highlyorchestrated developmental processes involving cell proliferation;migration, and differentiation. In each of the four transgenic lines,F1B-Tag tumors originated within the dorsal pons; specifically in thedorsal tegmental and/or pontine raphe nuclei. The histogenesis of theseclosely associated regions has been described in detail (Taber-Pierce,E. (1966) Histogenesis of the nuclei griseum pontis, corporispontobulbaris and reticularis tegmenti ponti (Bechterew) in the mouse.J. Comp. Neurol. 126, 219-240). Although a small number of cells in thedorsal tegmental nuclei appear to originate in the rhombic lip, thegenesis of the great majority of tegmental precursors occurs ongestation days 12-13 in the ependyma of the fourth ventricle. Theseprimitive cells are located within a limited region of the basal plate,just lateral to the midline, at the level of the pontine flexure.Similarly, neuroblasts giving rise to the pontine raphe, a narrow,midline structure dorsal to, and abutting, the dorsal tegmental nuclei,arise slightly earlier, on gestation days 10 and 11, from the sameventricular region (Taber-Pierce, E. (1966) Histogenesis of the nucleigriseum pontis, corporis pontobulbaris and reticularis tegmenti ponti(Bechterew) in the mouse. J. Comp. Neurol. 126, 219-240). Within 24-48hours of birth, neuroblasts destined for both regions migrate to theiradult position on either side of the midline, ventral to the mediallongitudinal fasciculus. Epigenetic influences during any of theseembryonic processes may be sufficient to block normal cellulardevelopment and lead to the formation of primitive neuroectodermaltumors at a later stage (Yachnis, A. T., Rorke, L. B., and Trojanowski,J. Q. (1994). Cerebellar dysplasia in humans: Development and possiblerelationship to glial and primitive neuroectodermal tumors of thecerebellar vermis. J. Neuropathol Exp. Neurol. 53:61-71).

The commitment of CNS progenitor cells to neuronal or glial lineage, andthe maturation of neurons and glia, are signaled by the coordinatedexpression of many developmentally regulated proteins (Anderson, D. J.(1999). Lineages and transcription factors in the specification ofvertebrate primary sensory neurons. Curr. Opinion. Neurobiol. 9,517-524). For example, vimentin is expressed in progenitor cells thatretain the plasticity to differentiate into cells of neuronal or gliallineage; it is replaced by synaptophysin and neuron-specific enolase inmature neurons, and by GFAP in astrocytes (Pekny, M., Johansson, C. B.,Eliasson, C., Stakeberg, J., Wallen, A., Perlmann, T., Lendahl, U.,Betsholtz, C., Berthold, C. H., Frisen, J. (1999). Abnormal reaction tocentral nervous system injury in mice lacking glial fibrillary acidicprotein and vimentin. J. Cell Biol. 145, 503-514). Three differentintermediate filaments, nestin, vimentin, and GFAP, are found inastrocytes and their precursors. In immature astrocytes, nestin andvimentin are the main intermediate filaments, whereas maturing and adultastrocytes contain vimentin and GFAP, respectively. During reactivegliosis, nestin production is resumed, and vimentin and GFAP expressionis upregulated in activated astrocytes (Eng, L. F., and Ghirnikar, R. S.(1994). GFAP and astrogliosis. Brain Pathol. 4, 229-237; Frisen, J.,Johansson, C. B., Torok, C., Risling, M., and Lendahl, U. (1995). Rapid,widespread, and longlasting induction of nestin contributes to thegeneration of glial scar tissue after CNS injury. J. Cell Biol. 131,453-464).

Comparison with widely used tumor markers suggests that the F1B-Tagtumor falls into the general category of the primitive neuroectodermaltumor (PNET). Our F1B-Tag mice comprises tumors which are at a uniquesite and at a unique stage of differentiation. The cells of the F1B-Tagtumors do not express any of the neuronal markers tested, includingsynaptophysin and neuron-specific enolase (FIG. 8). The cells of theF1B-Tag tumors express vimentin, a protein which is absent from anoligodendroglioma or choroid plexus papilloma (Reifenberger, G., Szymas,J., and Wechsler, W. (1987). Differential expression of glial- andneuronal-associated antigens in human tumors of the central andperipheral nervous system. Acta Neuropathol. 74: 105-123). The FIB-Tagtumor is unlikely to be an astrocytoma as the mature tumor does notexpress GFAP. The cells of the FIB-Tag tumors do not progress along theneuronal lineage to the point of expressing either glial or neuronalmarkers. It is believed that the cells of the FIB-Tag tumors areprohibited from reaching terminal differentiation, setting the stage fortumorigenesis, and providing a unique in vivo system in which to studythe induction and progression of PNETs .

The immunophenotype of the cells of the FIB-Tag tumors is consistentwith the tumor being at an early stage of differentiation. Therefore,the present transgenic mice are a valuable tool for the study oftumorigenesis, as well as the replenishment and differentiation ofneural stem cells.

Neural Stem Cells and Origin of Tumor Cells

Until recently, it was believed that adult brains were doomed to aconstant, steady decline. It appeared that, shortly after birth, neuronslost their ability to grow and cells that died could not be replaced.However, in the past few years, it has been reported that certain kindsof neurons can grow in adult brains (Temple, S., and Alvarez-Buylla, A.(1999). Stem cells in the adult mammalian central nervous system. Curr.Opin. Neurobiol. 9, 135-141). Self-renewing, totipotent embryonic stemcells may provide a virtually unlimited donor source for transplantation(Brustle, O., Jones, K. N., Learish, R. D., Karrarn, K., Choudhary, K.,Wiestler, O. D., Duncan, I. D., and McKay, R. D. G. (1999). Embryonicstem cell-derived glial precursors: A source of myelinating transplants.Science 285, 754-756). Recently, two groups have identified glial cellsin the dentate gyrus of the hippocampus as a source of proliferatingneurons. However, it is not clear whether the glial cells were derivedfrom the ependymal layer (Johansson, C. B., Momma, S., Clarke, D. L.,Risling, M., Lendahl, U., and Fris6n, J. (1999). Identification of aneural stem cell in the adult mammalian central nervous system. Cell 96,25-34) or the subventricular zone (Doetsch, F., Caille, I., Lim, D. A.,Garcia-Verdugo, J. M., and Alvarez-Buylla, A. (1999). Subventricularzone astrocytes are neural stem cells in the adult mammalian brain. Cell97, 703716) of the lateral ventricles. Although these precursor cellswere conveniently defined as glial cells because they expressed GFAP, itis quite possible that these GFAP-positive cells were precursors of bothglial and neuronal lineages (Barres, B. A. (1999). A new role for glia:Generation of neurons! Cell 97, 667-670). The resolution of thiscontroversy will require identification of additional markers thatidentify specific precursor cells. Our tumor cells, which lack both GFAPand synaptophysin, are expected to provide a useful source forgenerating these neural stem cell markers.

EXAMPLE 3 Preparation and Characterization of Tumor Cells Obtained fromMice of Example 2

Brain tumors dissected from 4-month old F1B-Tag mice were trituratedwith 22-gauge needles and grown in Dulbecco's modified Eagle medium(DMEM) supplemented with 5% fetal calf serum, EGF (10 ng/ml) and FGF1(10 ng/ml) as described (1 a). Aliquots of fresh growthfactor-containing medium were added every other day. Cells from a secondor third passage were used for differentiation studies. The resultsshowed that F1B-Tag tumor cells could differentiate into GFAP-expressingastrocytes when FGF1 and EGF were withdrawn from medium and LIF (10ng/ml) was added to medium for 6 days. These GFAP-expressing astrocytesare most likely derived from the F1B-Tag tumor as they continued toexpress FGF1 and T antigen. When the F1B-Tag cells were cultured in thepresence of (PDGF; 10 ng/ml), 75 to 95% of the cells were positive forthe immunostaining of synaptophysin. In the presence of thyroid hormoneT3 (10 mM), 25% to 45% of the cells became positive forgalactocerebroside (Ga1C), a marker for oligodendrocytes. The resultsshowed that the F1B-Tag cells are capable of differentiating into allthree neural progeny lineages. The fact that these tumor cells candifferentiate into astrocytes, neurons, and oligodendrocytes is evidencethat the F1B-Tag tumor cells are pluripotent neural stem cells.

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1. A DNA construct comprising a human FGF1B promoter as set forth innucleotide 43 to nucleotide 550 of SEQ ID NO: 2 operably linked to anucleotide sequence encoding the SV40 large T antigen.
 2. A DNAconstruct comprising a human FGF1B promoter as set forth in nucleotide10 to nucleotide 580 of SEQ ID NO: 2 operably linked to a nucleotidesequence encoding the SV40 large T antigen.
 3. The DNA construct ofclaim 1 wherein the SV40 large T antigen encoding sequence comprises anintron within said sequence.
 4. A DNA construct for providing neuralstem cells, said construct comprising the mouse FGF1B promoter operablylinked to a sequence encoding the SV40 large T antigen, wherein thesequence of the mouse FGF1B promoter is set forth in SEQ ID NO.
 3. 5. ADNA construct consisting of a human FGF1B promoter as set forth innucleotide 43 to nucleotide 550 of SEQ ID NO: 2 operably linked to anucleotide sequence encoding the SV40 large T antigen.
 6. A DNAconstruct consisting of a human FGF1B promoter as set forth innucleotide 10 to nucleotide 580 of SEQ ID NO: 2 operably linked to anucleotide sequence encoding the SV40 large T antigen.